The passage of virtually every molecule across the cell membrane is mediated by a class of proteins called transporters. Transporters are vital to the biology of all cells and a variety of diseases occur when these processes are perturbed or disrupted, as in several genetic disorders or the up-regulation of multidrug resistance transporters by tumor cells. The availability of high resolution structures of human transporters are essential to define the molecular structural basis of their mechanisms. We propose to establish a center for membrane protein structure determination, TransportPDB, with the objective of developing a comprehensive and efficient approach for pursuing the high-resolution x-ray crystal structures of 521 transporters in 48 families presently identified in humans and other targets from PSI-biology centers. For this purpose and to address the objectives of the PSI, we have the following specific aims: A1. An efficient pipeline will be established based on proven technologies and using our experience successfully crystallizing and solving the x-ray structures of integral membrane proteins. This pipeline will be based on several key principles: (a) target prioritization based on disease relevance and completing the protein-fold space coverage of human transporters, (b) the exclusive use of eukaryotic expression systems (Pichia pastoris and 293S mammalian cells) that have proven to deliver functional protein suitable for crystallization, (c) the cloning of constructs based on synthetic genes optimized for expression in both expression systems, and (d) using state-of-the-art data collection techniques for modestly diffracting crystals. The funnel-like organization will enable screening hundreds of human transporter targets and their close mammalian orthologs, driving towards the goal of successfully obtaining their x-ray crystal structures. A2. High-throughput methods and technology will be developed for functional and biophysical characterization of targets to rapidly identify conditions that maintain protein stability and function leading towards higher quality and better diffracting human transporter crystals. New crystal mounting methods, together with micro-beam/rastering technology and increased sensitivity in data collection (PILATUS detector), will be implemented that could be decisive for modestly diffracting membrane protein crystals. A3. Establish a resource for structural and functional data and other materials useful to the scientific community, including x-ray crystal structures of human transporters, codon-optimized clones, detergent solubilization conditions and corresponding stability properties of each target and homology models.
Transporters are vital to the biology of all cells and a variety of diseases occur when these processes are perturbed or disrupted, as in several genetic disorders or the up-regulation of multidrug resistance transporters by tumor cells. The development of effective therapeutics for the treatment of transport related diseases will be greatly accelerated by the availability of high resolution structures of human transporters, which are essential to understand the molecular structural basis of their mechanisms in detail.
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